This project has several components: 1. We are using NMR spectroscopy to characterize proteins required for the functioning of the human immunodeficiency virus. During the past year, we began a program for the preparation of isotopically labeled reverse transcriptase, the protein required for the synthesis of cellular DNA from the viral RNA. The use of isotopic labels will allow us to probe positions in the enzyme important for activity and drug interactions. 2. We have recently developed an NMR approach for the analysis of ligand binding to macromolecules. These studies look for pairs of ligands which bind to a macromolecular target at nearby sites. By selecting ligands which bind near to each other, it is anticipated that it will be possible to design more potent inhibitors by chemically combining these ligands. We will be applying this approach to the development of reverse transcriptase inhibitors. 3. Patients with a compromised immune system are more susceptible to adventitious bacterial infections, leading to increased dependence on antibiotic treatments. One mechanism of bacterial resistance to anti-folate drug therapy involves the expression of a plasmid type II dihydrofolate reductase, which is structurally unrelated to chromosomal DHFR and is resistant to bacterial antifolates such as trimethoprim. We have been studying the interaction of the type II DHFR with ligands in order to understand the catalytic mechanism and to develop new inhibitors. Inter-ligand Overhaser effects connecting the pyridine nucleotide and folate protons were observed and are being used to define a structure of the ternary complex.